The present invention relates to a method for making sphingomyelins and to the product produced thereby. More particularly, the present invention relates to a method for making optically pure D-erythro-sphingomyelins which may be labeled for identification and testing purposes.
Sphingomyelin is a naturally occurring composition found in most biological plasma membranes. As is known, sphingomyelin (N-acyl sphingosine-1-phosphocholine or ceramide-1-phosphocholine) consists of three components, namely, sphingosine, a fatty acid and phosphorylcholine. The structure of naturally occurring sphingosine may be defined as trans-D-erythro-2-amino-4-octadecene-1,3-diol or (2S,3R,4E)-2-amino-4-octadecene-1,3-diol and has the structural formula ##STR1## When the amino group of sphingosine is linked to a fatty acid, the formed compound is referred to as a ceramide and has the structural formula ##STR2## wherein R is an alkyl long-chain group of a fatty acid which typically has from fifteen to twenty-four carbon atoms. The third part of sphingomyelin is a phosphorylcholine group which is linked to the primary alcohol of ceramide. The phosphorylcholine group of sphingomyelin has the structural formula ##STR3## The phosphorylcholine is attached at the primary alcohol group as shown by the structural formula of sphingomyelin ##STR4## wherein R is an alkyl group of a long-chain fatty acid. Sphingomyelins which have a structural formula as illustrated are commonly found in biological plasma membranes of mammals.
As is well known, cells are characterized by a cytoplasmic membrane commonly referred to as a plasma membrane. This membrane creates a physical barrier by encapsulating the cytoplasm and providing internal compartments in which biological functions are carried out. This physical barrier created by the plasma membranes is necessary for the survival of the cell, such that the membrane excludes harmful substances, permits the acquisition of nutrients and energy and allows for the disposal of toxic materials from within the cell. In contrast to prokaryotic cells, eukaryotic cells include numerous intracellular organelles of widely different structures and functions, each bounded by its own membrane. Sphingomyelins are found in both plasma membranes and organelles of most mammalian tissues.
As is well known in the art, the essential structural unit of a biological membrane is the lipid molecule found in a bilayer arrangement. Within a biological membrane, these lipid molecules are interspersed with proteins which partially or completely traverse the bilayer via hydrophobic interactions, thus producing a mosaic of proteins and lipids. It has been shown that the most common lipids found in biological membranes are phospholipids. Particularly, phospholipids are essential for biological plasma membranes commonly found in the nervous tissue of all animal species. Sphingomyelin is one of those phospholipids.
Sphingomyelins have a high commercial value, especially in scientific research, medical studies, diagnostic testing and in the cosmetic industry. Some scientific research is focused on the regulation of sphingomyelin metabolism in biological systems. Many research laboratories study the influence of lipids on the activity of biological membranes and biological membrane-bound proteins. Much of this work is directed toward studying the dynamics of drug delivery, especially across the membranes of blood and nervous systems. Sphingomyelins have also gained great attention in brain and aging studies. Furthermore, several diseases, such as Niemann-Pick, atherosclerosis, cancer and some genetically transmitted diseases have been associated with abnormalities in sphingomyelin metabolism.
Thus, isolating and identifying sphingomyelins would be extremely beneficial when conducting medical studies and diagnostic testing relating to these diseases. However, naturally occurring sphingomyelins are heterogeneous in that they contain a multitude of sphingomyelins each having a variety of fatty acid chains attached thereto. Furthermore, naturally occurring sphingomyelins also contain small amounts of differing backbone long-chain bases, such as dihydrosphingosine and 4-hydroxyshinganine. Because of the heterogeneous character of naturally occurring sphingomyelins, it is extremely difficult to successfully conduct the desired scientific studies which require optically pure homogeneous sphingomyelins. Accordingly, it would be extremely desirable to have a method for producing homogeneous sphingomyelins that are optically pure. The most desirable sphingomyelins for these purposes may be referred to as D-erythro-sphingomyelins.
In addition to those qualities, it would also be desirable to have D-erythro-sphingomyelins having the ability of being labeled. Such labeled D-erythro-sphingomyelins are most suitable for medical and diagnostic testing since a label, such as an isotope, may have either radioactive or magnetic properties and thus be easily identified by conventional laboratory testing instruments. In the past, methods directed at producing optically pure D-erythro-sphingomyelins required many steps and resulted in extremely low overall yields. These methods provide very time-consuming and costly means for producing D-erythro-sphingomyelins.
For example, Shapiro et al, Journal of the American Chemical Society, 84, 1047 (1962), reported a 14 step procedure commencing from myristaldehyde to D-erythro-sphingomyelin having an overall yield of 0.1%. In another example, Bruzik, Journal of the Chemical Society, Chemical Communication, 329 (1986), discloses a 13 step procedure with an undisclosed overall yield. In addition to the undesirable lengthy procedures disclosed by Shapiro et al and Bruzik, both procedures require an additional optical resolution step to separate the resulting diastereomers. This additional separation step increases the number of steps required to produce the desired D-erythro-sphingomyelins. Several other attempts including semi-synthesis approaches have been made at producing sphingomyelins but these attempts have been unsuccessful with regard to the desired high yields and optical purity. A method for producing optically pure sphingomyelins having a high overall yield is extremely desirable in view of the expensive materials required to synthesize sphingomyelins.
Accordingly, there remains a need in the art for a method for making D-erythro-sphingomyelins that results in a high overall yield and permits the introduction of isotopes so as to form an isotopically labeled D-erythro-sphingomyelins useful in conducting scientific studies.